耐氟康唑白假丝酵母靶酶和外排泵编码基因过表达检测

Detection of overexpression of genes encoding target enzyme and efflux pumps in fluconazole-resistant Candida albicans

目的研究临床氟康唑耐药白假丝酵母中耐药相关基因MDR1、CDR1、CDR2和ERG11的表达水平及其与耐药的关系。方法采用Real-Time PCR技术检测耐药相关基因MDR1、CDR1、CDR2和ERG11的表达水平,计算敏感组菌株基因△Ct的平均值(x珋)与标准差(s),低于x珋-3 s为过表达。罗丹明6G(Rhodamine6G)外排实验检测外排泵高表达的白假丝酵母菌株中的外排功能。结果在12株耐药株中,4个基因中以CDR1和CDR2表达上调最为显著,分别有8株和3株,而ERG11只有1株表达上调,未见MDR1表达上调。外排实验中外排泵表达升高的菌株外排能力强,加入葡萄糖之后有些菌株外排进一步增强,而敏感菌株加入葡萄糖之前几乎无外排,加入葡萄糖之后略有外排。结论本实验基因表达与外排泵功能检测结果均证实,白假丝酵母对氟康唑耐药与ABC转运蛋白超家族过表达有关。

Objective To investigate the expression of MDRI, CDR1, CDR2 and ERGll genes in clinical fluconazole-resistant Candida albicans isolates, and explore its relationship with drug resistance. Methods The expression of drug resistance related genes MDR1, CDR1, CDR2 and ERGll was detected by Real-Time PCR, and mean （^-x） and standard deviation （s） of △Ct in genes of susceptible strains were calculated, with lower than ^-x - 3 s as overexpression. The efflux of Rhodamine6G was determined to examine the function of efflux pumps in the overexpression strains of Candida albicans. Results Among the 12 drug resistant strains, CDR1 and CDR2 were the most notable genes, with overexpression in 8 and 3 strains respectively. There was one strain overexpressing ERGll, and none for MDR1. The efflux of Rhodamine6G increased in the strains overexpressing efflux pumps, and the effect was enhanced after addition of glucose. As to the susceptible strains, there was almost no efflux before addition of glucose, and there was a slight change after addition of glucose. Conclusion Fluconazole-resistanee is related to the overexpression of ABC transport protein superfamily, which is confirmed by the gene expression profile and function test of efflux pumps.